Most secretory and membrane proteins are N-glycosylated and this modification often has profound effects on their stability and function. In fact, it is clear that in a number of human disorders protein glycosylation is altered. Although we now have a good understanding of the basic mechanism of N-glycosylation, the factors that control the subsequent folding of the newly formed N-linked glycoproteins into their final, stable three dimensional structure are not well understood. In addition, we only have a partial picture of how glycoproteins that do not fold correctly are catabolized. We will study disulfide bond formation and folding of glycoproteins, as well as the catabolism of unfolded glycoproteins, in the simple eukaryote, S. cerevisiae, because of the ease in which this organism can be genetically manipulated. With respect to disulfide bond formation during protein folding in the endoplasmic reticulum (ER), we will study the mechanism of an enzyme present in the lumen of the ER, protein disulfide isomerase (PDI). This enzyme catalyzes both the oxidation of thiols to form disulfide bonds and the isomerization of these disulfide bonds. In addition, PDI serves as a chaperone. We have prepared a collection of site specific cysteine to serine mutants, as well as a set of C-terminal deletions of PDI, and will use these in in vitro and in vivo experiments to better understand the mechanism by which PDI facilitates protein folding and functions in oxidation and disulfide bond isomerization. In the case of newly synthesized glycoproteins that do not fold correctly in the ER, it has been shown in higher eukaryotes that these misfolded proteins are exported out of the ER into the cytosol and degraded; the mechanism for their catabolism is being actively studied. In yeast this disposal process is less well understood, especially with respect to the fate of glycans on glycoproteins. A recently discovered soluble enzyme, PNGase, that deglycosylates glycoproteins may play a key role in this process in yeast. Therefore yeast PNGase will be cloned and sequenced. Then, in a series of in vivo experiments the possible function of PNGase in the catabolism of malfolded proteins will be investigated. In addition, the enzyme will be studied in vitro with respect to substrate specificity. Since both PDI inside the ER and PNGase in the cytosol interact with unfolded proteins, a clear understanding of both of these enzymes will provide new insights into factors regulating glycoprotein folding and catabolism.